Lyme Disease Spirochete Research Articles

Human microglia polarization following infection with the Lyme disease spirochete.

Infection with Borrelia burgdorferi can spread and cause central nervous system involvement, known as neuroborreliosis. Microglia phagocytose bacteria, mediate inflammation, and elicit an immune response toward the spirochete. Like other tissue macrophages, microglia can polarize into two different modulatory phenotypes, M1 and M2. We explored human microglial polarization changes upon infection with B. burgdorferi. HMC3 human microglia cell line was infected with B. burgdorferi for 24 h. Expression of polarization markers was evaluated via flow cytometry at 4 and 24 h. Secreted immunological mediators were evaluated using a multiplex ELISA system at 4, 18, and 24 h. An early decrease followed by a later increase in expression of M1 polarization marker iNOS was observed at 4 h, and 24 h, respectively. A decrease in M2 marker CX3CR1 occurred at 24 h. There were no changes in expression of M1 markers CD14, or in M2 markers CD163 and CD206. Multiplex ELISA evidenced an increase in secretion of activation markers MIP-1α, MIP- 1β, IP-10, chemotactic factor MCP-1, M1 polarization cytokine IL-8, and VEGF, at 4, 18, and 24 h. A decrease of iNOS at 4 h of infection suggests a diminished production of reactive nitrogen species that are a critical component of innate defense against infection. Increased iNOS and simultaneously decreased expression of CX3CR1 at 24 h, may suggest initiation of neuroprotective regulation of microglia recruitment to neuroinflammation. The dynamics of major inflammatory cytokines appear to be important in the microglial response to B. burgdorferi and should be further studied as these could become therapeutic targets in neuroborreliosis.

Development of novel multi-protein chimeric immunogens that protect against infection with the Lyme disease agent, Borreliella burgdorferi.

Lyme disease is the most common tick-borne disease in North America. A vaccine for use in humans is not available. Here, we detail the development of two chimeric vaccine antigens, BAF and Chv2M. BAF elicits Abs that target proteins and protein variants produced by Borreliella species in ticks (OspB and OspA) and mammals (FtlA/B). OspB serves as the backbone structure for the BAF chimeric. Two OspA221-240 epitope-containing domain (ECD) variants (#A1 and #A15) were inserted into a loop in OspB. The N-terminal region of the FtlA protein was joined to the C-terminus of the chimeric. The second chimeric, Chv2M, consists of L5 (loop 5) and H5 (helix 5) ECDs derived from diverse OspC proteins. Borreliella species produce OspC upon exposure to the bloodmeal and during early infection in mammals. Here, we demonstrate that BAF and Chv2M are potent immunogens that elicit Abs that bind to each component protein (FtlA, FtlB, OspB, and multiple OspA and OspC variants). Anti-BAF and anti-Chv2M Abs kill Borreliella burgdorferi strains through Ab-mediated complement-dependent and complement-independent mechanisms. Eighty percent (32/40) of mice that received a three-dose vaccine regimen were protected from infection with B. burgdorferi B31. Efficacy increased to 90% (18/20) when the amount of Chv2M was increased in the third vaccine dose. Readouts for infection were flaB PCR and seroconversion to VlsE. This study establishes proof of principle for a chimeric immunogen vaccine formulation that elicits Abs to multiple targets on the B. burgdorferi cell surface produced during tick and mammalian stages of the enzootic cycle.IMPORTANCELyme disease is a growing public health threat across parts of the Northern Hemisphere. Regions that can support sustained tick populations are expanding, and the incidence of tick-borne diseases is increasing. In light of the increasing risk of Lyme disease, effective preventive strategies are needed. Most vaccine development efforts have focused on outer surface protein A, a Borreliella burgdorferi protein produced only in ticks. Herein, we describe the development of a novel vaccine formulation consisting of two multivalent chimeric proteins that are immunogenic and elicit antibodies with bactericidal activity that target several cell surface proteins produced by the Lyme disease spirochetes in feeding ticks and mammals. In a broader sense, this study advances efforts to develop custom-designed vaccinogens comprised of epitope-containing domains from multiple proteins.

Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1.

Lyme borreliosis is caused by Borrelia burgdorferi and related bacteria. It is the most common tick-transmitted illness in the Northern Hemisphere. The ability of this pathogen to spread to a wide variety of locations results in a diverse set of clinical manisfestations, yet little is known regarding vascular escape of the spirochete, an important pathway for dissemination. Our current work has studied the traversal of B. burgdorferi across a monolayer of microvascular endothelial cells grown in culture. We show that this occurs by passage of the spirochetes directly through these cells rather than at cellular junctions and that internalization of B. burgdorferi is an intermediate step in the transmigration process. We also identify the first two host proteins, Cdc42 and Rac1, that are used by the spirochetes to promote traversal of the cellular monolayer. Our new experimental system also provides a new avenue for further studies of this important process.

Tick saliva interference with LTβR signaling facilitates the transmission of Lyme disease spirochetes

Tick saliva interference with LTβR signaling facilitates the transmission of Lyme disease spirochetes

DnaA modulates the gene expression and morphology of the Lyme disease spirochete.

Lyme disease is the most prevalent tick-borne infection in the Northern Hemisphere. Borrelia burgdorferi , the causative spirochete bacteria, has been maintained in nature for millennia in a consistent enzootic cycle between Ixodes ticks and various small vertebrate hosts. During the tick's blood meal, B. burgdorferi substantially increases its replication rate, alters its repertoire of outer surface proteins, and disseminates into the new vertebrate host. Across eubacteria, DnaA is the master regulatory protein that initiates chromosomal replication and acts as a transcription factor to regulate specific pathways. Here, we describe the roles that B. burgdorferi DnaA has on the physiology and gene expression of this medically important pathogen.

Comparative reservoir competence of Peromyscus leucopus, C57BL/6J, and C3H/HeN for Borrelia burgdorferi B31.

Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. Although laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability of these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here, we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models-C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi was able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle.IMPORTANCEA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host-usually rodents or birds-and ticks. In order to be successful in that endeavor, the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work, we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appears to colonize its natural host less successfully than conventional laboratory mouse models, which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.

Bacterial reprogramming of tick metabolism impacts vector fitness and susceptibility to infection.

Arthropod-borne pathogens are responsible for hundreds of millions of infections in humans each year. The blacklegged tick, Ixodes scapularis, is the predominant arthropod vector in the United States and is responsible for transmitting several human pathogens, including the Lyme disease spirochete Borrelia burgdorferi and the obligate intracellular rickettsial bacterium Anaplasma phagocytophilum, which causes human granulocytic anaplasmosis. However, tick metabolic response to microbes and whether metabolite allocation occurs upon infection remain unknown. Here we investigated metabolic reprogramming in the tick ectoparasite I. scapularis and determined that the rickettsial bacterium A. phagocytophilum and the spirochete B. burgdorferi induced glycolysis in tick cells. Surprisingly, the endosymbiont Rickettsia buchneri had a minimal effect on bioenergetics. An unbiased metabolomics approach following A. phagocytophilum infection of tick cells showed alterations in carbohydrate, lipid, nucleotide and protein metabolism, including elevated levels of the pleiotropic metabolite β-aminoisobutyric acid. We manipulated the expression of genes associated with β-aminoisobutyric acid metabolism in I. scapularis, resulting in feeding impairment, diminished survival and reduced bacterial acquisition post haematophagy. Collectively, we discovered that metabolic reprogramming affects interspecies relationships and fitness in the clinically relevant tick I. scapularis.

Structure analysis of the telomere resolvase from the Lyme disease spirochete Borrelia garinii reveals functional divergence of its C-terminal domain.

Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two of the most common tick-borne illnesses. A characteristic feature of these spirochetes is their highly segmented genomes which consists of a linear chromosome and a mixture of up to approximately 24 linear and circular extrachromosomal plasmids. The complexity of this genomic arrangement requires multiple strategies for efficient replication and partitioning during cell division, including the generation of hairpin ends found on linear replicons mediated by the essential enzyme ResT, a telomere resolvase. Using an integrative structural biology approach employing advanced modelling, circular dichroism, X-ray crystallography and small-angle X-ray scattering, we have generated high resolution structural data on ResT from B. garinii. Our data provides the first high-resolution structures of ResT from Borrelia spirochetes and revealed active site positioning in the catalytic domain. We also demonstrate that the C-terminal domain of ResT is required for both transesterification steps of telomere resolution, and is a requirement for DNA binding, distinguishing ResT from other telomere resolvases from phage and bacteria. These results advance our understanding of the molecular function of this essential enzyme involved in genome maintenance in Borrelia pathogens.

An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp/protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Evaluation of landscaping and vegetation management to suppress host-seeking Ixodes scapularis (Ixodida: Ixodidae) nymphs on residential properties in Connecticut, USA.

Ticks and tick-borne diseases are of increasing concern across the United States, particularly in the Northeast. Ixodes scapularis Say (Ixodida: Ixodidae) remains the primary vector for the Lyme disease spirochete, Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner). Prior studies established that I. scapularis can be found in greatest abundance in the 1-m forested ecotone surrounding the lawn edge in residential backyards. Our study was conducted on 42 properties in Guilford, CT, and sought to expand upon this premise by determining which key habitat features were associated with increased densities of host-seeking I. scapularis nymphs. We quantified nymphal abundances in 19 different habitat types that were posited to influence densities. We determined that nymphal I. scapularis densities were greatest in forested areas closest to lawn edges with leaf litter or understory vegetation present, as well as short lawns adjacent to woodland edges. Additionally, we determined that there were no significant declines in nymphal I. scapularis density where leaf litter was removed, lawns were left unmowed, or woodchip barriers were installed. Bird feeders and woodpiles were not associated with increased nymphal I. scapularis densities. However, areas adjacent to stone walls did have nearly 3 times the density of I. scapularis nymphs present compared with habitats without stone walls. The culmination of the results from this study can be utilized to create more targeted acaricide applications rather than broadcast spraying, as well as increase homeowner awareness for areas with heightened risk for exposure to nymphal I. scapularis, which are deemed the most epidemiologically important species and stage for pathogen transfer to humans.

Effects of outer surface lipoproteins on the nanomechanical properties of Lyme borrelia

The Lyme disease spirochete Borrelia burgdorferi causes an infection with diverse clinical outcomes, which can include arthritis as well as cardiac and neurological manifestations. B. burgdorferi expresses different outer surface lipoproteins at different stages in its infectious cycle, many of which are adhesins. Utilizing atomic force microscopy (AFM), we obtained topography images and force–distance curves of wild-type B. burgdorferi and mutant strains encoding different complements of surface lipoproteins. AFM data show that a reduced number of genome-encoded lipoproteins correlates with decreased binding probability, weakens unbinding force, and negatively affects cell elasticity.

295. Humanized Mice and Convalescent Humans Yield an Antibody Panel against the Highly Immunogenic Decorin Binding Protein A (DbpA) of the Lyme Disease Spirochete, Borrelia Burgdorferi

Abstract Background Lyme disease is the most commonly diagnosed tick-borne infection, affecting more than 300000 people in the United States annually. The disease is principally caused by the transmission of the spirochete bacteria, by the black-legged (deer) ticks. With a renewed call for interest in a Lyme disease vaccine, antibodies relevance continues due to their efficacy, safety and versatile nature. Humanized mice and humans recovering from the disease have successfully been used to generate therapeutic antibodies against Ebola and SARS-CoV-2 diseases. Methods Our study used humanized mice and convalescent patients to generate recombinant human antibodies that recognize decorin-binding protein A (DbpA), a lipoprotein that is proposed to function as spirochete adhesin. The mAbs expressed as human IgG-Fc fragments were characterized for reactivity with recombinant DbpA, native DbpA on the surface of live Borrelia burgdorferi, borreliacidal activity in the presence of human complement and, finally subject to epitope mapping using an array of methodologies. Results The predominant lineage of humanized mice antibodies utilized VH5-51, VH3-20, VH3-23 and VH3-53 all paired with VK1-33 families. Other common families included VH4-31 paired with VK1-39, VH4-4 and VH4-8 paired with VK-9. The human derived antibody utilized VH3-30 paired with VK1-33. We observed an overlap in the repertoire of kappa chains between humanized mice and human-derived mAbs. Conclusion Combining the humanized mice and human platforms enabled the identification of antibodies with a high binding and specificity to DbpA, which have potential prophylactic and therapeutic properties. Disclosures All Authors: No reported disclosures

The highly improved genome of Ixodes scapularis with X and Y pseudochromosomes.

Ixodes scapularis, the black-legged tick, is the principal vector of the Lyme disease spirochete, Borrelia burgdorferi, and is responsible for most of the ∼470,000 estimated Lyme disease cases annually in the USA. Ixodes scapularis can transmit six additional pathogens of human health significance. Because of its medical importance, I. scapularis was the first tick genome to be sequenced and annotated. However, the first assembly, I. scapularis Wikel (IscaW), was highly fragmented because of the technical challenges posed by the long, repetitive genome sequences characteristic of arthropod genomes and the lack of long-read sequencing techniques. Although I. scapularis has emerged as a model for tick research because of the availability of new tools such as embryo injection and CRISPR-Cas9-mediated gene editing yet the lack of chromosome-scale scaffolds has slowed progress in tick biology and the development of tools for their control. Here we combine diverse technologies to produce the I. scapularis Gulia-Nuss (IscGN) genome assembly and gene set. We used DNA from eggs and male and female adult ticks and took advantage of Hi-C, PacBio HiFi sequencing, and Illumina short-read sequencing technologies to produce a chromosome-level assembly. In this work, we present the predicted pseudochromosomes consisting of 13 autosomes and the sex pseudochromosomes: X and Y, and a markedly improved genome annotation compared with the existing assemblies and annotations.

The In Vitro Antimicrobial Susceptibility of Borrelia burgdorferi sensu lato: Shedding Light on the Known Unknowns.

Human Lyme borreliosis (LB) represents a multisystem disorder that can progress in stages. The causative agents are transmitted by hard ticks of the Ixodes ricinus complex that have been infected with the spirochete Borrelia burgdorferi sensu lato. Today, LB is considered the most important human tick-borne illness in the Northern Hemisphere. The causative agent was identified and successfully isolated in 1982 and, shortly thereafter, antibiotic treatment was found to be safe and efficacious. Since then, various in vitro studies have been conducted in order to improve our knowledge of the activity of antimicrobial agents against B. burgdorferi s. l. The full spectrum of in vitro antibiotic susceptibility has still not been defined for some of the more recently developed compounds. Moreover, our current understanding of the in vitro interactions between B. burgdorferi s. l. and antimicrobial agents, and their possible mechanisms of resistance remains very limited and is largely based on in vitro susceptibility experiments on only a few isolates of Borrelia. Even less is known about the possible mechanisms of the in vitro persistence of spirochetes exposed to antimicrobial agents in the presence of human and animal cell lines. Only a relatively small number of laboratory studies and cell culture experiments have been conducted. This review summarizes what is and what is not known about the in vitro susceptibility of B. burgdorferi s. l. It aims to shed light on the known unknowns that continue to fuel current debates on possible treatment resistance and mechanisms of persistence of Lyme disease spirochetes in the presence of antimicrobial agents.

75 The molecular basis for C1s inhibition by Lyme disease spirochetes

75 The molecular basis for C1s inhibition by Lyme disease spirochetes

Choreography of Lyme Disease Spirochete Adhesins To Promote Vascular Escape.

The Lyme disease spirochete Borrelia burgdorferi sensu lato can cause a multitude of clinical manifestations because of its ability to disseminate into any organ system via migration through soft tissue, the lymphatic system, and the circulatory system. The latter is believed to constitute the predominant pathway for dissemination to distal sites from the inoculating tick bite. In spite of its importance, the hematogenous dissemination process remains largely uncharacterized, particularly due to difficulties studying this process in a living host and the lack of an in vitro system that recapitulates animal infection. In the current work, we provide the first information regarding the stage of the vascular transmigration pathway where three important adhesins function during invasion of mouse knee joint peripheral tissue from postcapillary venules. Using intravital imaging coupled with genetic experiments employing sequential double infection, we show a complex temporal choreography of P66, decorin binding proteins (DbpA/B), and outer surface protein C (OspC) at discrete steps along the pathway of vascular escape, underscoring the importance of B. burgdorferi adhesins in hematogenous dissemination in the mouse knee joint and the complexity of vascular transmigration by a disseminating pathogen. IMPORTANCE Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted by a bite from an infected tick. Disease development involves a complex series of host-pathogen interactions as well as dissemination of the infecting organisms to sites distal to the original tick bite. The predominant pathway for this is believed to be hematogenous dissemination. The mechanism by which the spirochetes escape circulation is unknown. Here, using intravital microscopy, where the Lyme spirochete can be observed in a living mouse, we have studied the stage in the vascular escape process where each of three surface adhesins functions to facilitate escape of the spirochete from postcapillary venules to invade mouse knee joint peripheral tissue. A complex pattern of involvement at various locations in the multistage process is described using a unique experimental approach that is applicable to other disseminating pathogens.